1. Field of the Invention
This invention relates to the field of oligonucleotide synthesis, and, more particularly, to methods of loading mononucleosides on a solid support.
2. Description of the Related Art
Since Zamecnik and Stephenson, Proc. Natl. Acad. Sci. USA 75, 280-284 (1978) first demonstrated virus replication inhibition by synthetic oligonucleotides, great interest has been generated in oligonucleotides as therapeutic agents. In recent years the development of oligonucleotides as therapeutic agents and as agents of gene expression modulation has gained great momentum. The greatest development has been in the use of so-called antisense oligonucleotides, which form Watson-Crick duplexes with target mRNAs. Agrawal, Trends in Biotechnology 10, 152-158 (1992), extensively reviews the development of antisense oligonucleotides as antiviral agents. See also Uhlmann and Peymann, Chem. Rev. 90, 543 (1990).
Various methods have been developed for the synthesis of oligonucleotides for such purposes. See generally, Methods in Molecular Biology, Vol. 20: Protocols for Oligonucleotides and Analogs (S. Agrawal, Ed., Humana Press, 1993); Oligonucleotides and Analogues: A Practical Approach (F. Eckstein, Ed., 1991); Uhlmann and Peyman, supra. Early synthetic approaches included phosphodiester and phosphotriester chemistries. Khorana et al., J. Molec. Biol. 72, 209 (1972) discloses phosphodiester chemistry for oligonucleotide synthesis. Reese, Tetrahedron Lett. 34, 3143-3179 (1978), discloses phosphotriester chemistry for synthesis of oligonucleotides and polynucleotides. These early approaches have largely given way to the more efficient phosphoramidite and H-phosphonate approaches to synthesis. Beaucage and Caruthers, Tetrahedron Lett. 22, 1859-1862 (1981), discloses the use of deoxynucleoside phosphoramidites in polynucleotide synthesis. Agrawal and Zamecnik, U.S. Pat. No. 5,149,798 (1992), discloses optimized synthesis of oligonucleotides by the H-phosphonate approach.
Both of these modern approaches have been used to synthesize oligonucleotides having a variety of modified internucleotide linkages. Agrawal and Goodchild, Tetrahedron Lett. 28, 3539-3542 (1987), teaches synthesis of oligonucleotide methylphosphonates using phosphoramidite chemistry. Connolly et al., Biochemistry 23, 3443 (1984), discloses synthesis of oligonucleotide phosphorothioates using phosphoramidite chemistry. Jager et al., Biochemistry 27, 7237 (1988), discloses synthesis of oligonucleotide phosphoramidates using phosphoramidite chemistry. Agrawal et al., Proc. Natl. Acad. Sci. USA 85, 7079-7083 (1988), discloses synthesis of oligonucleotide phosphoramidates and phosphorothioates using H-phosphonate chemistry.
Solid phase synthesis of oligonucleotides by the foregoing methods involves the same generalized protocol. Briefly, this approach comprises anchoring the 3'-most nucleoside to a solid support functionalized with amino and/or hydroxyl moieties and subsequently adding the additional nucleosides in stepwise fashion. Desired internucleoside linkages are formed between the 3' functional group of the incoming nucleoside and the 5' hydroxyl group of the 5'-most nucleoside of the nascent, support-bound oligonucleotide.
Oligonucleotide synthesis generally begins with coupling, or "loading," of the 3'-most nucleoside of the desired oligonucleotide to a functionalized solid phase support. A variety of solid supports and methods for their preparation are known in the art. E.g., Pon, "Solid-Phase Supports for Oligonucleotide Synthesis," in Methods in Molec. Biol., Vol. 20: Protocols for Oligonucleotides and Analogs, p. 465 (Agrawal, Ed., Humana Press, 1993). Generally, the functionalized support has a plurality of long chain alkyl amines (LCAA) on the surface that serve as sites for nucleoside coupling. Controlled pore glass (CPG) is the most widely used support. It consists of approximately 100-200 .mu.m beads with pores ranging from a few hundred to a few thousand angstroms.
CPG supports are generally loaded by attaching a nucleoside-3'-succinate to the support through the succinyl group via an amide bond. Early efforts used dicyclohexylcarbodiimide (DCC) to activate the nucleoside-3'-succinate. Activation was accomplished by converting the nucleoside-3'-succinate into the symmetrical anhydride (Sproat and Gait, in Oligonucleotide Synthesis: a Practical Approach p. 83-115 (Gait, Ed., IRL Press, 1984)), or esterifying with p-nitrophenol (Atkinson and Smith, id. at 35-81; Koster et al., Tetrahedron 40, 103-112 (1984)) or pentachlorophenol (Gough et al, Tetrahedron Lett. 22, 4177-4180 (1981); Klerzek, Biochem. 25, 7840-7846 (1986)).
Montserrat et al., Nucleosides & Nucleotides 12, 967 (1993) reported that DCC and 1-hydoxybenzotriazole (HOBT) (used in equimolar amounts in a 14:1 dichloromethane/dimethylformamide solvent) resulted in higher loading densities than when DCC was used alone.
The use of DCC suffers from a number of disadvantages, however. First, DCC is highly toxic. Second, loading was tedious and gave only moderate yields (50-75%). Third, the coupling reactions were lengthy, requiring 3-4 d to make the activated succinates and an additional 4-7 d to couple them to the CPG. And finally, loading values were quite variable, and optimum loading of 30-40 .mu.mol/g was not always obtained.
Pon et al., Biotechniques 6, 768-775 (1988), improved on this method by employing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (DEC). Using this reagent along with a catalytic amount of dimethylaminopyridine (DMAP) in triethylamine/pyridine, Pon et al. observed direct coupling of the nucleoside-3'-succinate to the support. DEC, a smaller, less rigid carbodiimide (compared to DCC) was found to give much better results--loadings of up to 50-60 .mu.mol/g could be obtained in 24 hours.
In an alternative procedure, Damha et al., Nucl. Acids Res. 18, 3813-3821 (1990), showed that loading could be accomplished by succinylating the LCAA of the solid support, thereby providing a carboxylic acid functional group, followed by direct attachment of a nucleoside by esterification with DEC and DMAP in pyridine.
Tong et al., J. Org. Chem 58, 2223 (1993), followed the approach of Damha et al., supra, and compared the efficiency of DCC, DEC, and DIC in loading 3' unprotected cytidine onto a succinylated CPG support under basic conditions. The reaction was conducted in the presence of DMAP in dry pyridine. Loadings of 22 (DCC), 18 (DEC), and 30 (DIC) .mu.mol/g were obtained.
A major drawback of the current methods for loading solid support is that the main solvent is pyridine. Pyridine is toxic, has an obnoxious odor, and, therefore, is a work place and environmental hazard. In addition, DEC is costly, particularly when used in a large, production scale syntheses. Consequently, improved methods of column loading for oligonucleotide synthesis are desirable.